High resolution optical microscopic imaging technologies can significantly impact the diagnosis, staging, and treatment of a number of prevalent cancers in the United States. Fluorescent two-photon (2P) microscopic imaging has great potential for high resolution cancer diagnosis that can be a complementary noninvasive technique to clinical histological staining. The long-term goal is to develop fully biocompatible, highly specific and highly bright 2P probes for diagnosing, staging, and treating cancers. The objectives of this project are 1) to validate a promising 2P cancer imaging probe, conjugated polymer nanoparticle (CPN), in vitro by establishing highly specific cancer detection and 2) to develop extremely bright CPNs that can be excited at the power levels of confocal microscopy. The rationale for the proposed research is that developing extremely bright and highly cancer-specific CPNs can allow use of CPNs in a confocal endomicroscopic imaging system for noninvasive cancer diagnosis in the future. The objectives will be achieved by pursuing three specific aims. 1) Validate the CPN-probe for cancer cell specific labeling in vitro. Cancer cells are known to overexpress certain receptors and take up particles coupled with the ligands preferentially. We will develop cancer cell specific CPNs by coupling ligands of cancer cell surface receptors to CPNs. 2) Prove specificity improvement via multi-color and multi-targeting labeling. Multi-color labeling through a multi-targeting approach using fluorophores or particles has been known to improve the fidelity of cancer specific imaging. We will validate specificity improvement by monitoring two CPN colors, which will target different receptors in cancer cells. 3) Determine factors to further increase 2P brightness. Although CPNs exhibit already impressive 2P characteristics, a further increase in the 2P brightness by a factor of 10 will allow using similar ranges of conventional confocal la- ser powers for excitation, resulting in more practical uses of 2P imaging in biomedical applications including endomicroscopy. We will evaluate improvement of 2P brightness by examining the through-space effects in CPNs fabricated from conjugated oligomers with well-defined electronic structures. The proposed approach is innovative, because it uses CPNs that are about 100-1,000 times brighter than conventional fluorophores, extremely photostable comparable to quantum dots, and nontoxic to long-term cell growth in a tissue model system. The proposed research is significant, because the highly specific and bright 2P CPNs can be promising new imaging materials for clinicians and pathologists to improve their diagnostic accuracy and efficiency.